IOP elevations bring on oxidative stress

Key Points

The investigative team used a model involving short-term elevation of hydrostatic pressure in RGC-5 cells (a rat retinal ganglion cell line with both similarities to and differences from human RGC lines), then analyzed the localization and level of oxidative stress. They also studied oxidative stress in a mouse model following acute IOP elevation and examined the recovery of pressure-treated cells. In a final step, they tested the ability of two antioxidant compounds to inhibit the damage caused by oxidative stress, said Quan Liu, MD, PhD, a postdoctoral fellow in the Hamilton Glaucoma Center and department of ophthalmology, University of California San Diego, La Jolla.

The finding that a single, brief, acute elevation of pressure induces significant oxidative stress in RGCs in vivo and in vitro suggests that this could be an underlying mechanism for pressure-induced neuron death in glaucoma as well as in other human pathologies, Dr. Liu said.

In the cell model experiments, Dr. Liu treated RGC-5 cell cultures with varying hydrostatic pressures (0, 30, 60, and 100 mm Hg) for 2 hours at 37° C in a customized pressure chamber containing 5% CO₂ mixed with air. The cells were then harvested immediately or incubated for recovery experiments.

Oxidative stress was analyzed using immunocystostaining and Western blot. Researchers evaluated two biomarkers considered to be good indicators of oxidative stress: 4-hydroxynonenal (HNE) adducts, a lipoxidation product, and heme oxygenase-1 (HO-1), a phase II detoxification protein.

Tests of the effect of hydrostatic pressure on the culture media showed that there were no significant differences in pH, pCO₂, or pO₂ between the pressure-treated and control RGC-5 culture samples. However, HO-1 expression and adducts of HNE both increased significantly in the pressure-treated RGC-5 cells compared with nonpressured control cells.

Mouse model

A similar experiment was conducted using a mouse model. IOP in anesthetized mice was elevated to 30, 60, or 100 mm Hg for 1 hour using a micro-needle attached to a fluid column. The retinas were collected for analysis immediately after the elevation period. As in the cell culture experiments, the levels of HNE adducts and HO-1 increased after acute IOP elevation, indicating oxidative stress, Dr. Liu said.

Those results demonstrate that oxidative damage starts within hours of either elevated hydrostatic pressure or elevated IOP, Dr. Liu said. In both the cell and mouse models, data showed a dose-dependent increase of up to five-fold in HNE-protein adducts and up to 2.5-fold in HO-1 expression.

Cell recovery

A related series of tests was performed to investigate whether the cells could recover from the oxidative damage induced by the hydrostatic pressure. The RGC-5 cells were subjected to 60 mm Hg pressure for 2 hours, then incubated at normal pressure for 0, 1, 3, 6, and 10 hours.

The results showed that HNE adducts accumulated for 10 hours after the pressure elevation, which represents a detrimental effect, Dr. Liu said. However, the HO-1 was tightly regulated in the RGC cells, producing a protective effect.

An appropriate balance between the detrimental and protective functions is essential for the proper function and survival of the neurons during and after periods of elevated pressure, Dr. Liu explained.